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Multi-Biomarker Assessment in Common Carp (Cyprinus carpio, Linnaeus

1758) Liver after Acute Chlorpyrifos Exposure

Article in Water · June 2020

DOI: 10.3390/w12061837

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 water
Article
Multi-Biomarker Assessment in Common Carp
(Cyprinus carpio, Linnaeus 1758) Liver after Acute
Chlorpyrifos Exposure
Stela Stoyanova 1 , Elenka Georgieva 1, *, Iliana Velcheva 2 , Ilia Iliev 3 , Tonka Vasileva 3 ,
Veselin Bivolarski 3 , Stoil Tomov 4 , Krisztián Nyeste 5,6 , László Antal 5, * and Vesela Yancheva 2
1 Department of Developmental Biology, Faculty of Biology, Plovdiv University, 4000 Plovdiv, Bulgaria;
stela.st@abv.bg
2 Department of Ecology and Environmental Conservation, Faculty of Biology, Plovdiv University,
4000 Plovdiv, Bulgaria; anivelcheva@abv.bg (I.V.); veselayancheva@yahoo.com (V.Y.)
3 Department of Biochemistry and Microbiology, Faculty of Biology, Plovdiv University, 4000 Plovdiv,
Bulgaria; ilievini@abv.bg (I.I.); tonika1@abv.bg (T.V.); vb_ski404@zohomail.eu (V.B.)
4 Department of Urology and General Medicine, Plovdiv Medical University, 4000 Plovdiv, Bulgaria;
stoiltomov57012@gmail.com
5 Department of Hydrobiology, University of Debrecen, 4032 Debrecen, Hungary;
nyeste.krisztian@science.unideb.hu
6 Pál Juhász-Nagy Doctoral School of Biology and Environmental Sciences, University of Debrecen,
4032 Debrecen, Hungary
* Correspondence: e_tomova@abv.bg (E.G.); antal.laszlo@science.unideb.hu (L.A.)




Received: 18 May 2020; Accepted: 24 June 2020; Published: 26 June 2020


Abstract: The excessive use of pesticides at different stages of crop production can pose a great
danger to the aquatic environment, and particularly to fish. The purpose of the present work
was to assess the negative effects of chlorpyrifos (CPF) on the liver histological architecture and
the activities of marker enzymes in common carp (Cyprinus carpio Linnaeus, 1758), by applying a
multi-biomarker technique. The tested insecticide is categorized as a priority pollutant in surface
waters in terms of Directive 2013/39/EU. The carps were exposed to different and environmentally
relevant CPF concentrations for 72 h (a short-term acute experiment). The results showed that the
tested insecticide alters the liver histological structure, causing degenerative lesions, such as granular
and vacuolar degeneration; necrobiotic alterations and necrosis, as well as changes in the circulatory
system. In addition, CPF induces changes in the enzymatic activity of lactate dehydrogenase
(LDH), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), cholinesterase (ChE),
glutathione peroxidase (GPx) and catalase (CAT). The results from such experimental set ups could
be successfully used in the legislation related to the protection of water bodies from contamination, in
areas with intensive application of plant protection products used in agricultural practices, and also
in implementing the Water Frame Directive by using multi-biomarker approaches.

Keywords: fish; liver; histology; enzymatic activity; pesticides

1. Introduction
Modern agricultural practices lead to the indiscriminate use of various pesticides
that will eventually enter the aquatic environment [1]. Chlorpyrifos (CPF, O,O-diethyl
O-3,5,6-trichloro-2-pyridyl-phosphorothioate) is a broad spectrum organophosphorus insecticide
(OP) applied to control agricultural and domestic pests [2,3]. In addition, CPF is one of the most widely
used agricultural insecticides throughout the world, accounting for 50% of global insecticidal use [4,5].

Water 2020, 12, 1837; doi:10.3390/w12061837 www.mdpi.com/journal/water

Water 2020, 12, 1837 2 of 19

There are many pathways by which CPF can be distributed throughout aquatic ecosystems [4].
CPF can enter to aquatic ecosystems through rainfall, runoff and air-drift [5]. Wood and Stark [6]
explain that areas treated with CPF can result in concentrations of up to 4.3 µg/L in watercourses and
lakes. For example, CPF is one of the most widely used organophosphorus insecticides in Argentina,
and it is reported that concentrations of CPF have reached 10.8 µg/L in the surface waters of Pampa
Ondulada, Argentina [7]. CPF usage has the potential to change the aquatic environment, affecting
the boundary of tolerance of the aquatic ecosystems. Furthermore, CPF is one of the most common
pesticides found in fishery products in China [8].
Pesticide resistance, as well as bioaccumulative and toxic characteristics, cause severe short- and
long-term effects, even in low concentrations, and most of these toxic compounds could also accumulate
significantly in aquatic organisms [9]. Among all species of aquatic organisms, fish are regarded as good
indicators for assessing the quality of aquatic ecosystems [10]. Acute toxicity tests involving fish species
have proven to be useful for ecotoxicological evaluation to assess the potential hazards of various
chemical contaminants, e.g., pesticides [11–13]. Common carp (Cyprinus carpio Linnaeus, 1758) is one
of the most widespread fish species in the temperate regions of the world [14]. Common carp is very
popular in aquaculture industries, due to several specific features e.g., its feeding habits, high growth
rate, reproduction in captivity, adaptation to commercial diets and density. Moreover, common carp is
relatively insensitive, and can survive and accumulate contaminants at heavily polluted sites, which is
why it is used as a freshwater bioindicator in environmental toxicology [15]. However, anthropogenic
activities and environmental pollutants can be harmful for the physiology of common carp [16–21].
CPF can cause acute poisoning, and is reported to be involved in multiple mechanisms which
cause hepatic dysfunction, genotoxicity, and neurobehavioral and neurochemical changes [3,22,23].
CPF can also inhibit the acetylcholinesterase enzyme causing accumulation of acetylcholine in the
cholinergic receptors of nervous systems [3]. According to Banaee [24], under conditions of stress,
fish need extra energy, thus lactate dehydrogenase (LDH) is then involved in energy production
via anaerobic metabolism. In addition, as stated by Guptha et al. [25] and Ghorpade et al. [26],
under conditions such as those caused by pesticides, the changes in aspartate aminotransferase (ASAT)
and alanine aminotransferase (ALAT) enzymatic activity stimulate the process of gluconeogenesis and
the mobilization of L-amino acids.
Furthermore, oxidative stress is a general molecular mechanism in OP-induced toxicity [27].
However, fish have several defensive mechanisms to neutralize the impact of reactive oxygen species
(ROS) caused by OP-induced toxicity [3]. These include various antioxidant defense enzymes, e.g.,
catalase (CAT) and glutathione peroxidase (GPx), whose activities can be decreased significantly
by CPF intoxication. ROS which are not neutralized by the antioxidant defense system can cause
changes in the fish organism at the cellular, tissue and organ level [28,29]. Therefore, in recent years,
considerable research has focused on pesticide induced oxidative stress as a possible mechanism of
toxicity [3,30].
The liver is an organ that is widely used in biomarker approach studies on fish health assessment,
and is associated with detoxification processes, due to its function, position and blood supply. It is
also one of the most affected organs in contaminated waters, but also plays an important role in
fish physiology, i.e., in anabolism (proteins, lipids, carbohydrates) and catabolism (glycogenolysis,
detoxification), and is a storage center for different substances, mainly glycogen [31]. In addition,
the parenchymal liver tissue in fish has important physiological functions, such as the detoxification of
chemical contaminants (e.g., pesticides) [32].
Histology is a non-specific, relatively low-cost and valuable ecological risk assessment method [33].
Furthermore, changes in the activity of antioxidant enzymes can also be considered a reliable biomarker
for toxic damage [34,35]. Generally, biochemical alterations occur before the presence of the pathological
changes in the tissue [36]. In addition, the inhibition of enzymatic activity is also used to indicate tissue
damage [37]. Pesticides can produce free oxygen radicals via several mechanisms, i.e., interference with
electronic transport of reactive intermediates, the depletion of non-enzymatic antioxidants, inactivation
Water 2020, 12, 1837 3 of 19

of antioxidant enzymes and membrane lipid peroxidation [38,39]. The activity of the enzymes varies
in the different tissues, being higher in organs with high oxidative potential (e.g., in the liver) [37].
Based on the excessive use of pesticides and their negative impact on the aquatic environment
(e.g., [40]), the main aim of the present study is to determine the effects of environmentally relevant and
decreasing CPF concentrations on common carp, which is one of the most common freshwater species
in aquaculture and an important test organism in toxicology. Therefore, for this purpose, we applied a
multi-biomarker approach to assess the negative effects of CPF. We observed the histological structure
and measured the enzymatic activity of lactate dehydrogenase (LDH), aspartate aminotransferase
(ASAT), alanine aminotransferase (ALAT), cholinesterase (ChE), glutathione peroxidase (GPx) and
catalase (CAT) in the liver of the tested fish species.

2. Materials and Methods

2.1. Chemicals
Chlorpyrifos (CPF, CAS: 2921-88-2) with a density of 0.7688 g/cm3 at 21.3 ◦ C and a purity of
99.5%, diluted in cyclohexane was purchased for the purpose of this study. Stock solution of 0.03 µg/L
(30 ppm) CPF was prepared. Thereafter, 30 µL of it was diluted in 970 µL cyclohexane (100 ppm)
and calculated for the tested concentrations relatively for 100 L aquaria. All other applied chemicals
for histological and biochemical analyses were of analytical grade and were obtained from MERCK
(Darmstadt, Germany). They were used as received, without any further purification.
CPF is a degradable pesticide and it undergoes breakdown in the environment as a result of
biological, chemical and physical forces. In all systems (soil, water, plants and animals), the major
pathway of degradation begins with cleavage of the phosphorus ester bond to yield the breakdown
product trichloropyridinol (TCPy) [9]. This first step is a detoxification, as TCPy has no insecticidal
activity, and is considered toxicologically insignificant by regulatory authorities. In soil and water,
TCPy is further degraded via microbial activity and sunlight to carbon dioxide and organic matter.
In animals, TCP may be excreted directly or following conjugation (bonding with other chemicals);
in plants TCP conjugates are stored [13].
The solubility of CPF is temperature dependent, but at 25 ◦ C, it is about 1.4 mg/L. In water, CPF
readily adsorbs to suspended sediment and bottom materials. Volatilization is probably the primary
route of loss of CPF from water. Volatility half-lives of 3.5 and 20 days have been estimated for pond
water [41]. Its change into other natural forms (biotransformation) is slow [37]. Research suggests that
this insecticide is unstable in water, and the rate at which it is hydrolyzed increases with temperature,
decreasing by 2.5 to 3-fold with each 10 ◦ C drop in temperature. The rate of hydrolysis is constant in
acidic to neutral waters, but increases in alkaline waters. In water at pH 7.0 and 25 ◦ C, it is estimated
to have a half-life of 35 to 78 days [37].

2.2. Animals and Treatments

Healthy common carp specimens (n = 200), with normal morphology and no outside alterations
were obtained from the Institute of Fisheries and Aquaculture, Bulgaria, where the conditions are
controlled and strictly followed on a daily basis. The common carp had approximately the same
size (mean total length 13.5 ± 2.5 (SD) cm; mean body mass 22.5 ± 3.5 (SD) g). After transportation,
they were placed in 100 L glass aquaria with chlorine free tap water to acclimatize for seven days.
The photoperiod was 12 h light and 12 h dark. During the acclimatization period, the fish were fed
with commercial pellets for Cyprinids (CarpCo Excellent Koi Grower, Helmond, The Netherlands), but
they were not fed 2 days prior to the experimental exposure [42].
After this adaptation period, the fish were divided into four tested groups (n = 15), including
a control group (chlorine free tap water) and were treated in static conditions for 72 h with nominal
and environmentally relevant concentrations of CPF [40]. The experimental set up was performed in
triplicate. The tested insecticide was added only at the beginning of the experiment and the water
Water 2020, 12, 1837 4 of 19

was not renewed, as explained in other studies [43–45]. In addition, the half-life of CPF in water
ranges from 3.5 to 20 days and 35 to 78 days, depending on the pH, as described above; therefore,
it was supposed that the concentrations of CPF would not decrease considerably during the present
experimental design of 72 h. No control with cyclohexane only was used, as its concentration was
extremely low and no effect was expected.
The tested decreasing CPF concentrations were based on EU legislation and made up as 50 and 30%
of the allowable annual average concentrations (AA-EQS) in surface waters (0.03 µg/L) [40,43]. Thus,
the applied insecticide concentrations were 0.03 µg/L, 0.015 µg/L and 0.009 µg/L. A multi-parameter
portable meter (MultiLine® Multi 3510 IDS, WTW-Xylem Analytics, Weilheim, Germany) was used,
in order to measure the basic physico-chemical characteristics of water, including its conductivity,
dissolved oxygen, pH and temperature. They were monitored three times at the 24th, 48th and
72nd h [46]. Directive 2010/63/EU regarding the protection of animals used for scientific purposes
was followed thoroughly; hence, it was ensured that the fish were sacrificed with minimum pain,
suffering and distress. For this purpose, an anesthetic overdose was applied (100 mg/L water
of Tricaine methanesulfonate (MS222) (Argent Chemical Laboratories, Redmond, WA, USA)) and,
thereafter, the fish were dissected, and the samples were collected for different subsequent analyses.
Furthermore, the common carps were dissected according to the international procedures given by
Rosseland et al. [47], and the methods were approved by the Ethics Committee at the Faculty of Biology,
Plovdiv University, Bulgaria (No. 4/10.09.2019). An additional clarification, which we must make,
is that the main distinguishing anatomical feature of common carp from other Cyprinid fish is the
presence of hepatopancreas, which is a mixture between a liver and a pancreas. For the purpose of this
study we adopted the term “liver”, which also appears in the scientific literature.

2.3. Histological Procedures

A standard histological method involving hematoxylin-eosin staining was followed [48].
All histological samples were prepared for light microscopy analysis [48]. For each fish, the liver was
sampled and fixed in 10% neutrally buffered formaldehyde for 24 h. The preserved samples were
rinsed in tap water, dehydrated in a series of solutions with increasing ethanol concentrations (70%,
80%, 85%, 96%, 100%, respectively). Then they were cleared in xylene, infiltrated with liquid paraffin
with a melting point of 54–56 ◦ C and finally embedded in paraffin wax. The tested organs from
each fish were sampled using a rotary microtome (Leica RM 2125 RTS, Leica Microsystems, Wetzlar,
Germany); sections of 5 µm were stained with hematoxylin-eosin (H&E) and investigated with a light
microscope (Leica DM 2000, Leica Microsystems, Wetzlar, Germany).

2.4. Semi-Quantitative Screening

The histological alterations were presented according to the semi-quantitative system described
by Bernet et al. [49], which we adopted for the purposes of the study, but also modified slightly.
According to Saraiva et al. [50], a five-degree (0–5) severity gradation scale (SGS), which represents
the severity of each alteration, was applied. In addition, the organ index values (Io ) (circulatory
disturbances (ILC ), regressive lesions (ILR ), progressive lesions (ILP ), and inflammation (ILI )) were
calculated, in order to classify the severity of the histological response using classes based on the
scoring scheme suggested by Zimmerli et al. [51]: Class I (index ≤ 10)—normal hepatic tissue structure
with slight histological alterations; Class II (index 11–20)—normal hepatic structure with moderate
histological alterations; Class III (index 21–30)—moderate modifications of normal hepatic tissue;
Class IV (index 31–40)—pronounced histological alterations of the liver; Class V (index N 40)—severe
histological alterations of the liver. We also aimed to study the prevalence of liver histological changes.
Therefore, this was calculated as the percentage occurrence within the total number of examined slides
(n = 10) per individual.
Water 2020, 12, 1837 5 of 19

2.5. Biochemical Analysis

The liver samples were quickly thawed on ice and homogenized using a PYREX™ Potter-Elvehjem
tissue grinder with PTFE pestle (Thermo Fisher Scientific, Waltham, MA, USA) in a chilled phosphate
(50 mM, 300 mM NaCl, pH = 7.4) buffer [52]. The protein levels were measured by the Bradford [53]
method with Coomassie Brilliant Blue G-250, using bovine serum albumin at absorbance of
595 nm, and presented as milligram protein per milliliter homogenate. The enzymatic activities
of lactate dehydrogenase (LDH, EC 1.1.1.27), aspartate aminotransferase (ASAT, EC 2.6.1.1), alanine
aminotransferase (ALAT, EC 2.6.1.2), cholinesterase (ChE, EC 3.1.1.8), glutathione peroxidase (GPx,
EC 1.11.1.9) and catalase (CAT, EC 1.11.1.6) were measured spectrophotometrically, using a Beckman
Coulter Spectrophotometer DU 800 (Beckman Coulter, Inc., Brea, CA, USA) at 25 ◦ C. The LDH activity
was analyzed according to Vassault [54]. The ASAT and ALAT activities were determined using
the method described by Reitman and Frankel [55]. The ChE activity was analyzed by the method
developed by Burtis-Ashwood [56]. The CAT and GPx activities were determined using the method
described by Wendel [57].

2.6. Statistical Tests

IBM SPPS Statistics for Windows (Version 20.0, IBM Corp., Armonk, NY, USA) and GraphPad
Prism 7 for Windows (GraphPad Software, San Diego, CA, USA) were used for statistical analysis of
the data. The histological index results, percentage prevalence and enzymatic activities were expressed
as averages. The normal distribution was tested with the Shapiro-Wilk test. The homogeneity of
variances was tested with the Levene’s test, respectively. The results were also analyzed for significance
of differences between the control and treated groups by one-way analysis of variance (ANOVA),
followed by Tukey’s test (means comparison). The significance of results was set at p < 0.001, 0.01
and 0.05.

3. Results

3.1. Water Quality Parameters

The basic physico-chemical properties, such as conductivity, dissolved oxygen, pH and
temperature, were kept relatively constant during the exposure period (Table 1) (see [40]). The control
results did not differ significantly (ANOVA, p > 0.05), compared to the tested aquaria.

Table 1. Basic water properties measured in three chlorpyrifos (CPF) test tanks and control, according
to Yancheva et al. [40].

Conductivity Dissolved Dissolved Oxygen

Test Tank pH T (◦ C)
(µS/cm) Oxygen (mg/L) Saturation (%)
Control 8.3 ± 0.5 16.5 ± 1.5 370 ±5.5 8.5 ± 1.5 86.99 ± 12.82
0.009 µg/L CPF 8.0 ± 0.5 17.3 ± 2.0 365 ± 3.5 8.3 ± 0.3 86.38 ± 3.02
0.015 µg/L CPF 7.5 ± 1.5 18.5 ± 0.5 410 ± 0.5 7.0 ± 0.5 74.67 ± 5.38
0.030 µg/L CPF 8.5 ± 0.5 19.0 ± 0.5 353.3 ± 0.3 9.1 ± 1.0 98.06 ± 10.55

3.2. Liver Histological Structure

The results from the conducted histological study are presented in Tables 2 and 3, as well as
Figures 1 and 2. In the control group, we observed relatively normal liver morphology (Table 2,
Figure 1A). The hepatic structure was characterized by compactly arranged hepatocytes disposed in a
simple layer aligned with sinusoids. The parenchyma itself was primarily composed of polyhedral
hepatocytes, typically with central nuclei, with densely stained chromatin margins and a prominent
nucleolus. We also observed the pancreatic mass, which was situated around the branches of hepatic
portal veins. In addition, the pancreatic mass consists of two parts, which are the exocrine and endocrine.
Exocrine cells are larger and elongated, while endocrine cells are smaller and round. The morphology
Water 2020, 12, 1837 6 of 19

of the exocrine cells shows that they are arranged in an acinus form with a distinct nucleus, while the
endocrine cells are scattered in between the hepatic portal veins and the exocrine pancreas. Moreover,
the venous blood entered the liver caudally from the intestine via the hepatic portal veins, and branches
into capillaries known as sinusoids; the sinusoids were lined with reticuloendothelial cells,6 which
Water 2020, 12, x FOR PEER REVIEW of 19
were
in turn surrounded by hepatocytes (see [58]).
pancreas.
With regardMoreover, the venous blood
to the regressive liver entered
lesionsthe liver caudally
(Table from thegranular
2), we found intestine via the hepatic
(Figures 1B and 2B)
portal veins, and branches into capillaries known as sinusoids;
and vacuolar degeneration (Figure 1D) in the highest degree of expression. As shown the sinusoids were lined with
in Table 2,
reticuloendothelial cells, which were in turn surrounded by hepatocytes (see [58]).
all concentrations induced the same level of granular degeneration. Moreover, vacuolar degeneration
With regard to the regressive liver lesions (Table 2), we found granular (Figures 1B and 2B) and
showedvacuolar
a tendency towards
degeneration an 1D)
(Figure increase
in the in the degree
highest degreeofofexpression.
expression with in
As shown theTable
increased
2, all CPF
concentrations. We did not find fatty degeneration in the hepatocytes (Tables
concentrations induced the same level of granular degeneration. Moreover, vacuolar degeneration 2 and 3). Necrobiotic
changes,showed
such as karyolysis,
a tendency karyorrhexis
towards an increaseand karyopyknosis
in the (Figurewith
degree of expression 2D)thewere foundCPF
increased mainly in
concentrations.
a mild form We did not
of expression. Atfind
thefatty degeneration
higher in the hepatocytes
CPF concentration, these (Tables 2 andwere
changes 3). Necrobiotic
expressed in a
changes, such as karyolysis, karyorrhexis and karyopyknosis (Figure 2D) were found mainly in a
moderate degree, which, in turn, demonstrates the highest degree of degenerative processes leading to
mild form of expression. At the higher CPF concentration, these changes were expressed in a
necrosis.moderate
In addition, we also found necrosis in a moderate degree at the highest CPF concentration
degree, which, in turn, demonstrates the highest degree of degenerative processes leading
(Figure 2C). We also observed
to necrosis. In addition, we structural
also found alterations
necrosis in in the interstitial
a moderate degree tissue in a mild
at the highest CPFdegree of expression.
concentration
With regard to the
(Figure 2C).progressive
We also observedalteration in thealterations
structural commonincarp the liver, we observed
interstitial tissue in ahypertrophy
mild degree of in a mild
degree atexpression.
the lowerWith CPF regard to the progressive
concentrations, while, atalteration
the highestin the common carp
concentration, liver,
this we observed
histological lesion was
expressedhypertrophy
in a moderate in a mild degree at the lower CPF concentrations, while, at the highest concentration,
degree.
this histological lesion was expressed in a moderate degree.
The changes that occurred in the circulatory system were expressed on the one hand, in hyperaemia
The changes that occurred in the circulatory system were expressed on the one hand, in
(see Figure 1C), the(see
hyperaemia grade of which
Figure 1C), thewas
gradedetermined
of which was bydetermined
the proposedby theassessment scale as moderate
proposed assessment scale as at the
higher tested
moderateconcentrations,
at the higher while, at the lowest concentration,
tested concentrations, this lesion
while, at the lowest was absent
concentration, (Tablewas
this lesion 2). On the
other hand, we(Table
absent found2).inflammation, which
On the other hand, wewas expressed
found as lymphocytic
inflammation, which wasproliferation in the parenchyma.
expressed as lymphocytic
proliferation
It was found only atinthe thehighest
parenchyma.
applied It was
CPFfound only at the highest
concentration to be atapplied
a mildCPF concentration
degree to be at
of expression.
a mild degree of expression.

Figure 1. Histopathological alterations in the liver of common carp after the exposure with decreasing
Figure 1. Histopathological alterations in the liver of common carp after the exposure with decreasing
CPF concentrations based on Directive 2013/39/EU of the European Parliament and of the Council.
CPF concentrations based on Directive 2013/39/EU of the European Parliament and of the Council.
(A) Normal histological structure of carp liver (scale bar 100 μm) hematoxylin-eosin (H&E); (B)
(A) Normal histological structure
granular degeneration in carpofliver
carpat liver
0.009 (scale bar(scale
μg/L CPF 100 µm) hematoxylin-eosin
bar 50 (H&E);in(B)
μm) H&E; (C) hyperaemia granular
carp
degeneration
liver at 0.015 μg/L CPF (scale bar 50 μm) H&E; (D) vacuolar degeneration in carp liver at 0.015 μg/L liver at
in carp liver at 0.009 µg/L CPF (scale bar 50 µm) H&E; (C) hyperaemia in carp
CPFCPF
0.015 µg/L (scale bar 20
(scale μm)
bar 50H&E.
µm) H&E; (D) vacuolar degeneration in carp liver at 0.015 µg/L CPF (scale
bar 20 µm) H&E.
Water 2020, 12, 1837 7 of 19

Water 2020, 12, x FOR PEER REVIEW 7 of 19

The indices of histological changes highlight the distribution frequency of alterations and the
state ofThe indices
damage of of
thehistological changes
studied organ. In highlight
addition,the
thedistribution frequency ofchanges
indices of histological alterations and the
showed that
state of damage of the studied organ. In addition, the indices of histological changes showed
the regressive lesions were the most severe alterations observed in the liver parenchyma at all tested that the
regressive lesions were the most severe alterations observed in the liver parenchyma at all tested
concentrations. Moreover, ILR was significantly higher (p < 0.05) than the other indexes (ILC , ILP , ILI ).
concentrations. Moreover, ILR was significantly higher (p < 0.05) than the other indexes (ILC, ILP, ILI). In
In terms of the percentage prevalence, we observed the highest percent of histological alteration at
terms of the percentage prevalence, we observed the highest percent of histological alteration at the
the highest CPF concentrations (Table 3). Unlike the regressive, progressive and circulatory changes,
highest CPF concentrations (Table 3). Unlike the regressive, progressive and circulatory changes,
which showed a higher prevalence, the inflammation disturbances were observed less frequently, and
which showed a higher prevalence, the inflammation disturbances were observed less frequently,
only at the highest CPF concentration (see Table 3). According to the overall organ index, it was found
and only at the highest CPF concentration (see Table 3). According to the overall organ index, it was
to be categorized
found as Class as
to be categorized III Class
(indexIII21–30)—moderate modifications
(index 21–30)—moderate of normal
modifications tissue tissue
of normal at the at
highest
the
CPF statistically higher (ANOVA F = 6.123, p <
highest CPF concentration; it was also statistically higher (ANOVA F = 6.123, p < 0.05), comparedorgan
concentration; it was also 0.05), compared to the to
index at the other two tested concentrations.
the organ index at the other two tested concentrations.

Figure
Figure 2. 2. Histopathologicalalterations
Histopathological alterationsininthe
the liver
liver of
of common
commoncarp carpafter
afterthe
theexposure
exposurewith decreasing
with decreasing
CPF concentrations based on Directive 2013/39/EU of the European Parliament
CPF concentrations based on Directive 2013/39/EU of the European Parliament and of the and of the Council
Council
(0.03 μg/L). (A) Initial process of lipid accumulation (scale bar 20 μm) H&E; (B) granular degeneration
(0.03 µg/L). (A) Initial process of lipid accumulation (scale bar 20 µm) H&E; (B) granular degeneration
(scale bar 50 μm) H&E; (C) necrosis (scale bar 50 μm) H&E; (D) nuclear alterations—karyolysis
(scale bar 50 µm) H&E; (C) necrosis (scale bar 50 µm) H&E; (D) nuclear alterations—karyolysis (arrow)
(arrow) and karyopyknosis (scale bar 50 μm) H&E.
and karyopyknosis (scale bar 50 µm) H&E.
Water 2020, 12, 1837 8 of 19

Table 2. Histopathological alterations in the liver of common carp after the exposure with decreasing CPF concentrations based on Directive 2013/39/EU of the
European Parliament and of the Council.

Functional Score Value Index for Each Group

Reaction Importance Factor
Unit of the Alteration
Pattern Control 0.009 µg/L 0.015 µg/L 0.03 µg/L (0.009; 0.015; 0.03 µg/L)
Tissue
Circulatory ILC = 0
Liver Hyperaemia WLC1 = 1 0 0 2 2
Disturbances * ILC = 2
Intercellular oedema 0 0 0 0 ILC = 2
Granular degeneration WLR1 = 1 1 2 2 2
Deposits (lipids) WLR3 = 1 0 0 0 0
Liver Nuclear alterations WLR4 = 2 0 0 1 2
Necrosis WLR5 = 3 0 0 1 2
ILR = 4
Regressive Vacuolar degeneration WLR6 = 2 0 1 2 3 ILR = 11
Lesions *
Architectural and ILR = 19
WLR7 = 1 0 0 0 1
structural alterations
Interstitial Deposits WLR8 = 1 0 0 0 0
tissue
Nuclear alterations WLR9 = 2 0 0 0 0
Necrosis WLR10 = 3 0 0 0 0
Liver Hypertrophy WLP1 = 1 0 1 1 2 ILP = 1
Progressive
Interstitial ILP = 1
Lesions * Hypertrophy WLP2 = 1 0 0 0 1
tissue ILP = 3

Activation of the
reticuloendothelial system WLI1 = 1 0 0 0 1 ILI = 0
Inflammation * Liver in the parenchyma (RES) ILI = 0
ILI = 3
Infiltration WLI2 = 2 0 0 0 1
Index organ IC = 1 I0.009 = 5 I0.015 = 14 I0.03 = 27
* According to Bernet et al. [49], Saraiva et al. [50] and Zimmerli et al. [51]: Reaction index (circulatory disturbances (ILC ), regressive lesions (ILR ), progressive lesions (ILP ) and inflammation
(ILI )) is calculated by the sum of multiplied importance factors and score values of the alterations of the corresponding reaction pattern. The sum of reaction indices of the organ is
equivalent to the organ index (Io). Organ index values (Io) for: circulatory disturbances (ILC ), regressive lesions (ILR ), progressive lesions (ILP ) and inflammation (ILI ) calculated in order to
classify the severity of the histological response. Index for each group shows the sum of the score value of the histological alterations corresponding to each reaction pattern at the different
CPF concentrations (0.009; 0.015; 0.03 µg/L). Importance factor (W) for: circulatory disturbances (ILC ), regressive lesions (ILR ), progressive lesions (ILP ) and inflammation (ILI ) shows how
big impact the histological alteration has on the fish; severity gradation scale (SGS): (0)—no histological alterations, which represented normal liver histological structure; (1)—mild
histological alterations; (2)—moderate histological alterations; (3)—pronounced histological alterations; (4)—severe histological alterations and (5)—very severe histological alterations.
Importance factor: (1)—minimal pathological importance, the lesion is easily reversible when the toxicant exposure stops; (2)—moderate pathological importance, the lesion is reversible in
most cases if the stressor is neutralized and (3)—marked pathological importance, the lesion is generally irreversible, leading to partial or total loss of the organ function.
Water 2020, 12, 1837 9 of 19

Table 3. Percentage of histopathological alterations in common carp liver after the exposure with
decreasing CPF concentrations based on Directive 2013/39/EU of the European Parliament and of
the Council.

Prevalence, %
Histopathological Alterations
Control 0.009 µg/L 0.015 µg/L 0.030 µg/L
Circulatory
Liver Hyperaemia 0 0 32 38
Disturbances
Intercellular oedema 0 0 0 0
Average, % 0 0 16 19
Granular degeneration 11 36 41 47
Deposits (lipids) 0 0 0 0

Liver Nuclear alterations 0 0 16 42

Necrosis 0 0 21 36

Regressive Vacuolar degeneration 0 17 36 47

Lesions Architectural and
0 0 0 14
structural alterations
Interstitial Deposits 0 0 0 0
tissue
Nuclear alterations 0 0 0 0
Necrosis 0 0 0 0
Average, % 1.2 5.9 12.7 20.7
Liver Hypertrophy 0 13 21 32
Progressive
Lesions Interstitial
Hypertrophy 0 0 0 12
tissue
Average, % 0 6.5 10.5 22
Activation of the
reticuloendothelial
0 0 0 25
Inflammation Liver system in the
parenchyma (RES)
Infiltration 0 0 0 37
Average, % 0 0 0 31

3.3. Liver Enzymatic Activity

We found an increase in the LDH specific activity with increasing insecticide concentrations.
However, the LDH specific activity differed significantly among the different groups (ANOVA F = 22.33;
p < 0.001) (Figure 3).
In addition, the statistical data showed that the ASAT specific activity differed significantly among
the different groups (ANOVA F = 8.008; p < 0.01). The ALAT specific activity also differed significantly
among the different groups (ANOVA F = 6.345; p < 0.01) (Figure 4).
Water 2020, 12, x FOR PEER REVIEW 10 of 19

9 a
Water 2020, 12, 1837 10 of 19
8 a

(units/mg protein)
Water 2020, 12, x FOR PEER REVIEW 10 of 19
7 a
69 a
58
a
protein)
47 a
activity

36
(units/mg

25
LDH activity LDH

14 b
03
2 control 0.009 μg/L 0.015 μg/L 0.030 μg/L
1 treatment
b
0
Figure 3. Lactate dehydrogenase
control (LDH)0.009
activity in common0.015
μg/L carp liver
μg/L under different CPF exposures.
0.030 μg/L
Bars represent means ± SD of the control and experimental groups. Different letters indicate
treatment
significant differences among treatments (p < 0.05).

Figure
In 3. Lactate
addition,
Figure thedehydrogenase
statistical data(LDH) activity
showed in common
that the ASATcarpspecific
liver
liver under different
activity
under CPF exposures.
differed
different significantly
exposures.
amongBars
Barstherepresent
different
represent means
groups
means ± SD
± SD of of control
(ANOVA
the the F
control
= 8.008;
and andp experimental
< 0.01).
experimental groups.specific
The ALAT
groups. Different Different letters
activity
letters indicate indicate
also differed
significant
significant
significantly
differences differences
among
amongthe among (p
different
treatments < 0.05). (ANOVA
treatments
groups (p < 0.05). F = 6.345; p < 0.01) (Figure 4).

In addition, the statistical data showed that the ASAT specific activity differed significantly
0.5
among the different groups (ANOVA F = 8.008; p < 0.01). The ALAT specific activity also differed
b
significantly among the different groups (ANOVA F = 6.345; p < 0.01) (Figure 4).
0.4
and ALAT activity
(units/mg protein)

0.5
0.3 b b
0.4
activity

0.2 a a
(units/mg protein)

a
ab
ASAT

0.3 b
ASAT and ALAT

0.1 a
a
0.2 a a
a
0 ab
control 0.009 μg/L 0.015 μg/L 0.030 μg/L
0.1 a
atreatment
0
Figure 4.4. Aspartate
Aspartate aminotransferase
aminotransferase
control (ASAT)
0.009(white
(ASAT) μg/L bars)
(white and alanine
bars)0.015 aminotransferase
0.030 μg/L (ALAT)
μg/L aminotransferase
activity (black
activity (blackbars)
bars)inincommon
commoncarpcarpliver
liverunder
underdifferent
different CPF
CPF
treatment exposures.
exposures. Bars
Bars represent
represent means
means ± SD
± SD of
the control
of the and experimental
control groups.
and experimental Different
groups. letters indicate
Different letters significant differencesdifferences
indicate significant among treatments
among
(p < 0.05). (p < 0.05).
treatments
Figure 4. Aspartate aminotransferase (ASAT) (white bars) and alanine aminotransferase (ALAT)
On
On one
activity hand,
one(black the
theChE
hand,bars) inChEspecific
common activity
carp
specific liver decreased
under
activity compared
different
decreased to thetocontrol
CPF exposures.
compared groupgroup
Barscontrol
the (Figure
represent means 5),
± SDwhich
(Figure 5),
confirmed
which the
of confirmed toxic
the control the potential
and toxic of the
experimental tested
groups.
potential of theinsecticide
Different in
tested lettersterms of
indicate
insecticide enzymatic
in significant inhibition.
terms of differences However,
enzymatic among
inhibition.
the enzymatic
treatments
However, activity
(p < 0.05).did
the enzymatic not differ
activity significantly
did not among the
differ significantly different
among groups (ANOVA
the different F = 2.073;
groups (ANOVA F
p= > 0.05).p > 0.05).
2.073;
On one hand, the ChE specific activity decreased compared to the control group (Figure 5),
which confirmed the toxic potential of the tested insecticide in terms of enzymatic inhibition.
However, the enzymatic activity did not differ significantly among the different groups (ANOVA F
= 2.073; p > 0.05).
Water 2020, 12, 1837 11 of 19
Water 2020, 12, x FOR PEER REVIEW 11 of 19
Water 2020, 12, x FOR PEER REVIEW 11 of 19

0.7
0.7
aa
protein)
(units/mgprotein) 0.6
0.6
0.5
0.5
activity(units/mg

0.4
0.4 aa
0.3
0.3 aa aa
ChEactivity

0.2
0.2
ChE

0.1
0.1
00
control
control 0.009
0.009 μg/L
μg/L 0.015
0.015 μg/L
μg/L 0.030
0.030 μg/L
μg/L
treatment
treatment

Figure
Figure 5.
Figure 5. Cholinesterase
5. Cholinesterase(ChE)
Cholinesterase (ChE)
(ChE) activity
activity in
in common
activity in common
common carp liver
carpcarp under
liverliver
under different
under CPF
different
different exposures.
CPF CPF Bars
exposures.
exposures. Bars
represent
Bars
represent means
represent
means ±
meansSD of the
± SD±ofSD control
theofcontrol and
the control experimental
and and groups;
experimental
experimental ‘a’
groups;
groups; letter represents
‘a’ letter
‘a’ letter that
represents
represents there
thatthat were
there
there were no
were
no
significant
no differences
significant
significant among
differences
differences among
among treatments
treatments
treatments (p >
(p (p > 0.05).
0.05).
> 0.05).

Similar
Similar to
to the
the ChE,
ChE, the
the activity
activity of
of GPx
GPx was
was reduced
reduced regarding
regarding the
the control
control and the
and the applied
the applied
applied
concentrations of CPF (Figure 6). The GPx specific activity differed significantly among the different
concentrations of CPF (Figure 6). The GPx specific activity differed significantly among the different
groups
groups (ANOVA
(ANOVAFF
groups (ANOVA F=== 25.33;
25.33; pp <
<< 0.001).
0.001).

0.07
0.07 aa
0.06 aa
protein)

0.06
(units/mgprotein)

0.05
0.05 bb
activity(units/mg

0.04
bb
0.04
0.03
0.03
GPxactivity

0.02
0.02
GPx

0.01
0.01
00
control
control 0.009
0.009 μg/L
μg/L 0.015
0.015 μg/L
μg/L 0.030
0.030 μg/L
μg/L
treatment
treatment

Figure
Figure 6.6.
6. Glutathione
Glutathione peroxidase
peroxidase (GPx)
(GPx) activity
activity in
in common
common carp
carp liver
liver under
under different CPF exposures.
Figure under different
different CPF exposures.
Bars
Bars represent
Bars represent means
representmeans
means ± SD
± SD
± SD of the control
of control
of the the control and experimental
and experimental
and experimental groups. Different
groups. Different
groups. Different letters
letters
letters indicate indicate
indicate
significant
significant differences
significant differences
differences among treatments
among (p
among treatments treatments (p < 0.05).
< 0.05). (p < 0.05).

On
On the
On the other
the other hand,
other hand, the
hand, the activity
the activity
activity ofof CAT
of CAT generally
CAT generally increased
generally increased proportionately
increased proportionately
proportionately toto the
to the increased
the increased
increased
insecticide
insecticide concentrations.
concentrations. However,
However, the the
CAT CAT specific
specific activity
activity differed
differed significantly
significantly
insecticide concentrations. However, the CAT specific activity differed significantly among among among
the the
different
the
different
groups groups
(ANOVA
different (ANOVA F = 39.88;
F = 39.88; Fp =<39.88;
groups (ANOVA p < 0.001)
0.001)p(Figure (Figure 7).
< 0.001)7).(Figure 7).
Water 2020, 12, 1837 12 of 19
Water 2020, 12, x FOR PEER REVIEW 12 of 19

40
d
35
CAT activity (units/mg protein) a
30
25
c
20
15
10 b
5
0
control 0.009 μg/L 0.015 μg/L 0.030 μg/L
treatment

Figure 7.
Figure 7. Catalase
Catalase (CAT) activity in
(CAT) activity in common
common carp
carp liver
liver under
under different CPF exposures.
different CPF exposures. Bars
Bars represent
represent
means ± SD of the control and experimental groups. Different letters indicate significant differences
means ± SD of the control and experimental groups. Different letters indicate significant differences
among treatments
among treatments (p (p <
< 0.05).
0.05).

Withregard
With regardto to
thethe enzyme
enzyme CAT,CAT,
a lowera activity
lower was
activity
foundwas found
at the lowestatapplied
the lowest applied CPF
CPF concentration,
concentration,
which we thinkwhich we athink
indicates moreindicates a more active
active involvement involvement
of GPx, of GPx, participate
as both enzymes as both enzymes
in the
participate in
mechanism of the mechanism
antioxidant of antioxidant
protection. protection.
In addition, In addition,
the CAT the CAT specific
specific enzymatic activity enzymatic
increased
activity
with theincreased
increasingwith the increasing
pesticide pesticideAt
concentrations. concentrations.
the highest CPFAt concentration,
the highest CPFtheconcentration, the
activity of CAT
activity
was of CAT
higher than was higher than
the control, which the control,indicates
probably which probably indicates
the occurrence of the occurrence
oxidative stress,ofdue
oxidative
to the
stress, due to the
accumulation accumulation
of free of freeinoxygen
oxygen radicals radicalsunder
the organism in theCPF
organism under CPF toxicity.
toxicity.

4. Discussion
4. Discussion
The
The water
water quality
quality parameters
parameters during
during the the experiment
experiment were were in in accordance
accordance with with FAOFAO (1999)
(1999) [59]
[59]
and
and APHA
APHA (2005)
(2005) [46],
[46], although
although there
there were
were differences
differencesamongamongthe theexperimental
experimentalgroups. groups.
The
The histological
histological changes
changesin infish
fishliver
livercould
couldserve
serveasasuseful
useful biomarkers
biomarkers that
thatshow
show thetheinfluence
influence of
toxic substances
of toxic substanceson the organism,
on the organism,duedue to the
to liver’s mainmain
the liver’s function
functionin detoxification
in detoxification lipophilicity [60].
lipophilicity
We agree with Xing et al. [8] and Deb and Das [61], who state that degeneration,
[60]. We agree with Xing et al. [8] and Deb and Das [61], who state that degeneration, pyknotic nuclei, pyknotic nuclei, fatty
infiltration or total
fatty infiltration ornecrosis at different
total necrosis levels oflevels
at different expression, as a result
of expression, as of pesticide
a result intoxication,
of pesticide could be
intoxication,
established in the liverinparenchyma.
could be established AccordingAccording
the liver parenchyma. to Bukharito etBukhari
al. [62], theet al.hepatic
[62], thelesions
hepaticare lesions
associatedare
with the increased toxicant concentrations, which we confirmed in our study.
associated with the increased toxicant concentrations, which we confirmed in our study. In addition, In addition, the production
of
thefree radicals,of
production lipid
freeperoxidation
radicals, lipidand changes inand
peroxidation thechanges
antioxidant in thestate are vitalstate
antioxidant factorsarefor thefactors
vital toxic
effects
for the of pesticides
toxic effects of onpesticides
the liver onhistological structure [63].
the liver histological According
structure to Albañil to
[63]. According Sánchez
AlbañiletSánchez
al. [45],
lipid degeneration
et al. [45], in fish liver
lipid degeneration caused
in fish liver by pesticides
caused leads to
by pesticides dysfunctions
leads to dysfunctions in theinmicrosomal
the microsomal and
mitochondrial
and mitochondrial systems of cell,
systems of which can lead
cell, which can to thetoinhibition
lead of theofsynthesis
the inhibition the synthesis of lipoproteins.
of lipoproteins.As a
result of both acute and chronic exposure to pesticides, the hepatic function
As a result of both acute and chronic exposure to pesticides, the hepatic function is affected, resulting is affected, resulting in a
reduction of protein concentration [64], which, in turn, affects the liver tissue.
in a reduction of protein concentration [64], which, in turn, affects the liver tissue. We support the We support the opinion
of Velisekofet Velisek
opinion al. [65], who
et al.highlighted
[65], who that the toxic effects
highlighted that the of pesticides
toxic effects could of lead to overproduction
pesticides could lead of to
lipid peroxidation in various tissues, as well as in the liver, which causes
overproduction of lipid peroxidation in various tissues, as well as in the liver, which causes histological alterations. In
addition,
histological reactive oxygen
alterations. Inspecies
addition,(ROS) induce
reactive peroxidative
oxygen damage
species (ROS) in theperoxidative
induce liver, and this could be
damage in
one of theand
the liver, molecular
this could mechanisms
be one of theof CPF toxicity,
molecular which we suggest
mechanisms needs further
of CPF toxicity, which detailed
we suggest research.
needs
We agree
further with Oruç
detailed and Üner
research. [66],with
We agree whoOruç stateandthatÜner
the physiological
[66], who statechanges that the in fish dependchanges
physiological on the
species
in fish specificity,
depend on thethechemical
speciescharacteristics
specificity, of thethechemical
pesticides,characteristics
their concentration of the andpesticides,
the duration of
their
the exposure period. According to Pereira Maduenho and Martinez [67],
concentration and the duration of the exposure period. According to Pereira Maduenho and Martinez the most severe histological
changes
[67], the occur with increased
most severe cell changes
histological and nuclear occurvolume, which iscell
with increased associated
and nuclear with volume,
cytoplasmic which andis
associated with cytoplasmic and nuclear alterations, and biliary dysfunction with increased
metabolic activity of the hepatocytes, which we confirmed in the present study. Similar to our study,
Water 2020, 12, 1837 13 of 19

nuclear alterations, and biliary dysfunction with increased metabolic activity of the hepatocytes, which
we confirmed in the present study. Similar to our study, Xing et al. [8] found varying degrees of
hydropic degeneration, vacuolization, pyknotic nuclei and fatty degeneration due to CPF, and atrazine
exposure at concentrations close to LC50 of the tested pesticides. In parallel, changes in the enzymatic
activity of CAT and GPx were also observed by the authors. Tilak et al. [68] observed similar alterations
in the liver of Gibelion catla (Hamilton 1822) under the action of CPF. The pathological lesions included
cytoplasm degeneration in the hepatocytes, atrophy, vacuolar degeneration, blood vessel destruction,
necrosis and damage in the hepatocyte cell membrane, and pyknotic hepatocyte nuclei, which we also
found in the liver of common carp exposed to CPF. Moreover, Mohamed [69] and Kostić et al. [70]
described that the stasis of blood could be responsible for cellular degeneration and necrosis in the liver.
In addition, sinusoidal congestion blocks blood from the hepatic artery and the interbiliary portal vein,
which has to pass through the sinusoids on its way to the central vein. We support the opinion of Devi
and Mishra [31], who consider that the parenchyma cytoplasmic and nuclear degeneration observed in
the liver, as well as the vacuolization of the hepatocytes, is probably due to metabolic damage related to
the exposure to CPF contaminated water. In our previous study [71], which involved glyphosate-based
herbicide, the histological lesions in common carp liver also showed a different degree of expression,
which increased in proportion to the increasing pesticide concentrations.
Overall, the observed histological changes in the common carp liver could be used as reliable
biomarkers, which indicate the negative effects of the exposure to toxic substances in the environment.
Although the liver is the primary organ of detoxification, the impact of pesticides such as CPF may
change the ecological plasticity of fish to stress factors of the environment. In addition, abnormal
hepatocytes can interfere with the normal metabolism, and thus disrupt fish health, or even cause
fish death. Similarly to Devi and Mishra [31], we consider the studies on histological changes
observed in fish liver, as well as in other tested organs (gills, spleen, kidney, muscle) to be an effective
biological tool for assessing the health of fish populations, which, in turn, reflect the health of the entire
aquatic ecosystem.
According to Banaee [24], Stoyanova et al. [71,72] and Yancheva et al. [52], LDH activity is usually
related to cellular metabolic activity, and serves as the major enzyme between glycolysis and the citric
acid cycle. LDH is an important enzyme, due to its function in the anaerobic pathway of energy
production. According to the authors mentioned above, fish, under conditions of stress, need extra
energy, thus, LDH is then involved in energy production via anaerobic metabolism. Our results suggest
changes in the processes of glycolysis and glycogenogenesis; therefore, we found a decreased LDH
activity at the lowest CPF concentration and an increased LDH activity at the other two, higher CPF
concentrations. In general, LDH activity is a commonly used diagnostic tool and demonstrates an
increase in anaerobic metabolism due to the depletion of energy and stress caused by environmental
changes and the effect of different toxicants. Furthermore, we agree with Banaee [24], according to
whom, alterations in the LDH activity are indicative for hepatic tissue damage, which serves as a
diagnostic tool in toxicological studies.
According to Guptha et al. [25], the transaminases are a group of enzymes that elevate the
activity of enzyme phosphorylase, which has a significant role in glycogenolysis, and also represents a
major link between protein and carbohydrate metabolisms. We agree with Ghorpade et al. [26] that
the increase in the activity of ALAT could be used as a more specific biomarker for acute toxicity,
compared to ASAT activity. Moreover, the changes in the ALAT levels play an important role in the
glucose-alanine cycle in the liver. Similar to the authors mentioned above, we found that, under
conditions of stress caused by the CPF exposure, the elevated levels of ASAT and ALAT stimulate the
process of gluconeogenesis and the mobilization of L-amino acids.
Overall, the most significant results from our study showed a decrease in the enzymatic activity of
LDH and ALAT compared to the control. This tendency was most clearly established at the lowest CPF
concentration. Thus, we suppose that an initial process of triglyceride accumulation in the hepatocytes
started. This is probably due to the increased pyruvate in the liver cells. Hence, via the pyruvate
Water 2020, 12, 1837 14 of 19

dehydrogenase complex, the amount of Acetyl-CoA also increases. Furthermore, Acetyl-CoA is used
for the synthesis of fatty acids and cholesterol. Therefore, the increased amount of fatty acids in the
liver leads to increased triglyceride synthesis and hyperlipidemia associated with lipid infiltration
in the hepatocytes [52,71,72]. Although we did not identify cells with typical fatty degeneration by
the applied histological method with H&E, morphological changes in the cytoplasm and the location
of the nucleus of single cells were determined, which probably suggests the beginning of the initial
process of lipogenesis (Figure 2A).
According to Payne et al. [73], ChE inhibition is used as a marker to prove contamination of
aquatic ecosystems with organophosphorus compounds. In our study, we also measured a decreased
ChE activity in the fish treated with CPF, compared to the control. This activity was in an inverse
relationship with the applied CPF concentrations, i.e., it was lower in the samples treated with higher
insecticide concentrations. Therefore, we also recommend the use of ChE in multi-biomarker studies
on the effects of pesticides on fish, and, more precisely, in risk assessment and monitoring programs.
In addition, according to Assis et al. [74], the interaction between cholinesterases (ChEs) and pesticides
lies behind the ability of the enzymes to signal inhibition processes several days or weeks after exposure,
even when in low concentrations. We agree with the authors who state that the in vitro approach could
gain more precision in the correlation between pesticide concentrations and the resulting inhibition.
We confirmed that changes in the activity of antioxidant enzymes in the fish organism could serve
as a biomarker for the toxicity of organic contaminants in water, as stated by Ahmad et al. [75]. Similar
to Yousafzai and Shakoori [76], we consider that the variations in the specific enzymatic activity in the
tested samples compared to the control presents an indicator of the effects of pesticides on fish health.
Oxidative stress due to pesticide effects can be used in the toxicological studies as a possible mechanism
for chemically induced toxicity [61,77,78]. Furthermore, induced oxidative stress by organophosphorus
substances causes depletion of mitochondrial energy (ATP), overuse of proteolytic enzymes and
DNA fragmentation. Biological systems have mechanisms to counteract free radical damage in the
organism. The main mechanisms include antioxidant enzymes defense, which neutralizes free radicals.
Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase
(GRx), vitamin C, vitamin E and beta-carotene represent the most important antioxidant protection in
biological systems [79]. In general, the biomarkers of oxidative stress can be divided into two main
groups: free radical biomarkers in biological systems and antioxidant protection factors [80,81]. In the
first group, Slaninova et al. [37] include the presence of products resulting from damage to free oxygen
radicals, i.e., the most commonly used products of lipid peroxidation. The biomarkers applied in
the present study belong to the group of antioxidant protection factors. Narra et al. [82] observed
a significant increase in CAT activity during a CPF exposure, while at the same time, the authors
established a decrease in the enzymatic levels during a recovery period. In the present study, we also
observed an increased activity of CAT at the highest applied insecticide concentration. This could
possibly be an indicator of oxidative stress caused by the applied CPF concentration, which equals
the annual average environmental quality standards (AA-EQS), according to the national and EU
legislation regarding the Water Frame Directive.
Overall, we think that, at the lower applied CPF concentrations, the enzyme GPx becomes
more active, while at the higher concentrations, it is CAT which is more active. Clasen et al. [83]
observed oxidative damage in carp, due to the toxic action of a complex of organic pollutants under
field conditions, and chronic exposure. The oxidative damage was expressed in an increase in lipid
peroxidation in the liver, which, in turn, is an indicator of oxidative stress. Similar to our study,
the authors observed an increase in the activity of CAT resulting from the toxic action of the applied
toxicants and the inclusion of the antioxidant protection of the organism. Nunes et al. [84] observed
alterations in the CAT enzymatic activity in common carp and zebra fish (Danio rerio Hamilton, 1822)
under different concentrations of cypermethrin and CPF for 96 h. The enzymatic activity in common
carp decreased compared to the control group, whereas that in zebra fish showed an increase compared
to the control. This in turn, confirms that the changes in the activity of the antioxidant enzymes
Water 2020, 12, 1837 15 of 19

depend on the concentrations of the applied toxicants, the time of exposure and the species specificity.
Similar to our study, Narra et al. [82] also found that the GPx activity was significantly decreased
during the CPF exposure. As stated by Guptha et al. [25], GPx alters the protection provided for
the cellular and subcellular membranes from peroxidative damage by eliminating H2O2. We agree
with Slaninova et al. [37], who stated that a consistent decrease in antioxidant enzymes is due to the
excessive generation of free radicals, most likely generated by CPF in our study.
We support the opinion of Yonar [85] that the decrease or increase in enzymatic activities can be
explained either by their consumption and induction during the conversion of free radicals into less
harmful or harmless metabolites, or, secondarily, by the direct inhibitory or stimulatory effect of ROS
that may be caused by CPF in the present study.

5. Conclusions
Overall, the results showed that CPF alters the liver histological structure, causing degenerative
and necrobiotic alterations, and necrosis, as well as changes in the circulatory system. In addition,
CPF induces changes in the enzymatic activity of LDH, ASAT, ALAT, ChE, GPx and CAT. In summary,
we can conclude that the use of multi-biological approaches in environmental monitoring is crucial
for assessing aquatic pollution and its impact on ecosystems. In addition, we consider that complex
experimental set ups, such as the present one, including histological and biochemical biomarkers,
should be performed to better determine the toxic impact of pesticides on fish. These results can be
used for the application of the EU Water Framework Directive for preventing and reducing aquatic
contamination with priority organic substances in Bulgaria and other developing countries in the
Balkans, as well as for implementing proper agricultural practices.

Author Contributions: Conceptualization, S.S., V.Y.; Methodology, S.S., V.Y., E.G., I.I., T.V., V.B.; Formal analysis,
K.N.; Supervision, I.V.; Writing—review and editing, S.S., S.T., K.N., L.A., V.Y.; Funding acquisition, S.S., E.G.,
L.A., V.Y. All authors have read and agreed to the published version of the manuscript.
Funding: The National program “Young Researches and Postdocs, 2018” financed by the Ministry of Education
and Science, Bulgaria, is highly appreciated. The authors also thank the Ministry for Education and Science,
Bulgaria and The Scientific Research Fund for the financial support for project M26/3 (Scientific Fundamental
Research for Young Scientists and Postdocs). Krisztián Nyeste was supported by the ÚNKP-20-4 New National
Excellence Program of the Ministry for Innovation and Technology in Hungary. The research was also supported
by the Higher Education Institutional Excellence Programme (NKFIH-1150-6/2019) of the Ministry of Innovation
and Technology in Hungary, within the framework of the 4th thematic programme of the University of Debrecen.
Acknowledgments: We also thank the experts at the Regional Accredited Laboratory, Executive Environment
Agency, Ministry of Environment and Water, Plovdiv, Bulgaria for providing the toxicant.
Conflicts of Interest: The authors declare no conflict of interest.

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